ES2284398A1 - Formulation of liposomal vesicles in aqueous solutions with tear film characteristics - Google Patents

Abstract

Formulation of liposomal vesicles in aqueous vehicles with tear film characteristics. The present invention addresses the preparation of a pharmaceutical liposomal system in an aqueous solution that incorporates a substance or polymer with mucomimetic and/or mucoadhesive properties and that, owing to its components and characteristics, can replace the precorneal film. This invention is applicable to the areas of pharmacy and medicine.

Description

Formulation of liposomal vesicles in aqueous solutions with tear film characteristics.

Object of the invention

The present invention relates to the formulation of liposomal vesicles in aqueous solutions with tear film characteristics. The present invention describes a liposome formulation in aqueous vehicles that contain mucin or mucin-like substances, substances mucomimetics or polymers with mucoadhesive properties that, at the corneal surface temperature, have characteristics similar to the precorneal film of the human eye. Bliss Preparation could be used instead of the film natural and as a medicinal preparation in some eye diseases as is the case with dry eye syndrome.

This invention fits within the areas of pharmacy and medicine.

State of the art

The ocular surface is known to be formed by conjunctival epithelium, corneal epithelium, glands accessory lachrymal and meibomian glands. That surface It is covered by a continuous film, with a thickness of approximately 10 µm, called precorneal film or film tear Until a few years ago the theoretical structuring, generally accepted, it included three types of components (lipid, sero-aqueous and mucinous) distributed in three layers: lipidic, aqueous and mucinosa (Ibrahim H, Buri P, Gurny R. Pharm Acta Helv 1988, 63: 146-53).

Recent studies consider the film precorneal is a structure formed by the components aqueous-protein and mucinous combined to form a hydrated gel In turn, this gel would be protected by a lipid film, whose components would be produced mainly by the meibomian glands and whose function would be prevent the evaporation of the tear and improve the stability of the tear film (Pflugfelder SC, Solomon A, Stern ME. Cornea 2000; 19 (5): 644-649. McCulley JP, Shine W. Tr Am Ophth Soc 1997; 95: 79-93).

According to the proposed model, the film Precorneal would consist of two phases:

- Hydrophilic polar phase, in contact with the aqueous-mucinous layer that is composed of phospholipids, sphingomyelin, ceramides and cerebrosides.

- Non-polar hydrophobic phase in contact with the atmosphere and composed of non-polar lipids such as esters of wax, cholesterol esters, triglycerides, free fatty acids and hydrocarbons

The phospholipid fraction represents approximately 1-5% of the total lipid secretion, with the highest concentration being phosphatidylcholine (FC) with a percentage close to 40% of the total phospholipids. Other phospholipids, such as phosphatidylethanolamine appear in a percentage of 18%, the remaining ones (a total of 10) being in a range between 3 and 9%. Probably, this fraction produces a decrease in tension
surface of the aqueous phase facilitating the extensibility of the precomeal film during the flickering movement.

The usual treatment of dry eye is to relieve symptoms by applying tear substitutes topically. The typical composition of these preparations includes polymeric solutions such as the one collected in US, patent No. 4,973,580 (Babiole) in which the ophthalmic formulation includes hyaluronic acid using hydrogen peroxide as a preservative. Formulations are also described in which tear film-like components are provided as hypotonic solutions of lecithin including cellulose derived viscosifying agents as it appears in US Patent No. 4,421,748 (Trager). The use of phospholipids for the treatment of dry eye appears in various patents. Emulsion-like systems containing positively charged phospholipids are described as described in the following: US Patent No. 4,914,088 (1990) (Korb :); 5,278,151 (1994) (Korb :); 5,371,108 (1994) (Korb :), 5,294,607 (1994) (Korb :). Positively charged liposomes are also described US patent No. 4,804,539 (Guo) (1989) and US Patent No. 4,818,537 (Guo) in which positively charged liposomes are used which are suspended in aqueous solutions containing high viscosity polymers such as hydroxyethylcellulose, methylcellulose, hydroxypropylcellulose and vinyl derivatives such as polyvinylpyrrolidone, polyvinyl alcohol and mixtures thereof. They are also collected
emulsions containing phospholipids, non-polar oils and emulsifiers such as US Patent No. 6,656,460 (Benite).

In none of these patents does the use of neutral or negatively charged liposomes that destabilize at the temperature of the precorneal film or that associated with mucin or with mucoadhesive substances or similar to mucin or mucomimetics as is the case of the invention that described below.

Description of the invention

The method object of the invention herein describe refers to the preparation of a formulation pharmaceutical that acts as a substitute for the precorneal film. The formulation incorporates phospholipid liposomal vesicles as hydrophilic polar phase and non-polar lipids, both vehiculized in aqueous solutions containing mucin or substances with Mucin-like properties or mucomimetic substances or mucoadhesive polymers. The most relevant advantages of this invention consist of the use of phosphatidylcholine whose transition temperature is lower than the temperature of the surface of the cornea and also incorporates polymers or substances mucoadhesive and / or mucomimetic (mucin or polymers such as acid hyaluronic, cellulose derivatives, chondroitin sulfate, chitosan, colominic acid, thiol derivatives or other component Similary).

The components of the formulation and specifically the phospholipids that make up the liposomes will allow the formation, on the corneal surface, after the destabilization of the liposomal vesicles, of an insoluble monomolecular film in water that acts preventing the evaporation of the aqueous phase and, In addition, the surface tension of the latter will decrease which favors its rapid extensibility. Liposomes are prepared with phosphatidylcholine obtained from soy lecithin as major component, cholesterol and α-tocopherol. Phosphatidylcholine contains acyl residues of unsaturated fatty acids presenting a transition temperature lower than surface temperature of the cornea, which guarantees the rapid formation of the film on the aqueous phase, once applied on the surface of the cornea. Cholesterol, meanwhile, stabilizes this film by reduce the fluidity of the matrix formed by the remains polyunsaturated phosphatidylcholine. Finally the α-tocopherol ensures the chemical stability of the double bonds avoiding the possible peroxidation.

Liposomes are transported in aqueous solution which contains an isotonizing agent (trehalose, sodium chloride, glucose ...) to get the appropriate osmolaridac according to its use clinical. The solutions may be isotonic, or hypotonic, One once formed the liposomes are incorporated into aqueous solutions that contain one or more substances or polymers with characteristics mucoadhesive or mucomimetic in order to produce a increase in the residence time of the formulation and of the components of the destabilized liposomes on the surface ocular. Thus the maintenance of the new film is favored a once formed and prevents aqueous evaporation of the surface corneal The concentrations of this last component will depend of the desired final viscosity in the formulation, of its interaction with mucin, its surface tension and behavior rheological expected after administration. Also included proteins in the formulation in order to promote stability of the film formed and improve its lubricating properties. These proteins are found in natural tears and can be α-macroglobulin, lysozyme, lipocalin and lactoferrin

To this formulation can be added different compounds, mostly film components natural tear, which improve the formation characteristics and permanence of the precorneal film and / or acting as re-epithelializing, anti-inflammatory and surface antioxidants ocular, and / or favoring corneal epithelial differentiation and conjunctival Within these substances are:

Mucoadhesive polymers such as acid hyaluronic, cellulose derivatives, chondroitin sulfate, chitosan, colominic acid, thiol derivatives (or other component Similary).

Neutral lipids and low lipids polarity such as waxes, cholesterol esters, triglycerides, acids Free fatty and hydrocarbons.

Vitamin A.

sodium, potassium, calcium, chlorine and ions baking soda.

Vitamin C.

Albumin or pre-albumin

Immunoglobulin A (IGA).

epithelial growth factor (epithelial growth factor: EGF).

Transforming growth factor Beta (Beta transforming growth factor) (TGF-?).

Fibroblast growth factor acid (Acidic fibroblast growth factor) (aFGF).

Fibroblast growth factor basic. (Basic fibroblast growth factor) (bFGF).

Antiproteases such as macroglobulin

? Neural factors such as substance P and insulinlike growth factor.

Antibacterial agents such as Ig G, lysozyme and complement.

Long chain fatty acids such as gadoleic, palmitic, palmitoleic, stearic, oleic acid, linoleic, arachidic, linolenic, eicosenic, lignoceric, lactic and mystical.

Hydrophilic lipids such as phospholipids, sphingomyelin, ceramides and cerebrosides.

Embodiment of the invention

The present invention which relates to the formulation of liposomal vesicles in aqueous solutions with tear film characteristics, further illustrated through the following examples, which are not limiting of its scope, which is defined by the claim attached.

The liposomal vesicles object of the present Invention were performed according to the classic Bangham method. For this dissolved phosphatidylcholine, cholesterol and α-tocopherol (in different proportions) in chloroform obtaining a final concentration of phosphatidylcholine 8 mg / ml. Once saturated this solution with nitrogen is introduced into the rotary evaporator flask at a temperature of 30-35ºC with a moderate vacuum. After evaporation a thin lipid film formed on the walls of the solvent which then hydrated. The hydration phase was performed with a saturated aqueous solution of nitrogen containing the isotonizing agent at a temperature of 37 ° C, using pearls of glass that produced shear formation by shear multilamellar The final phosphatidylcholine concentration is adjusted according to the volume of isotonizing vehicle.

After two hours of rest and in the absence of light, the sonication of the dispersion was carried out maintaining the Product temperature between 5 and 10ºC with crushed ice. The preparation ended by making 5 passes of the dispersion by 0.8 µm filters.

The mucin or mucoadhesive substance and / or Mucomimetic was added by diluting the liposomes to the concentration desired end The final concentrations of liposomes in the Polymer solution can range from 1 mg / ml to 40 mg / ml.

The rest of the possible components are added, based on its physicochemical characteristics, with the isotonizing agent or with the mucomimetic substances.

The base liposomes were prepared from phosphatidylcholine from soybeans, and cholesterol (8: 1) and reconstituted with water and hypotonic sodium chloride solutions. The influence of the sonication process on the final size of the vesicles was studied by comparing the use of an ultrasound probe for 2.5 min. and an ultrasonic bath for 15 min. (Figure 1). The yield of the process of preparation of the lipid vesicles, in both cases, was greater than 90%.

Dispersions of liposomes in water at a FC concentration of 20 mg / ml showed pH values between 6.9 and 7.2. The average particle diameters for the different batches prepared with ultrasonic bath they oscillated between 392 and 478 nm. The percentage of higher particles at 1 µm it was, in all cases, less than 2%.

Surface tension measurements were made with solutions of different concentrations of liposomes, obtaining the data in Figure 2.

Cell viability tests were performed with hypotonic aqueous solutions of base liposomes and base liposomes with vitamin E in macrophage cell cultures. For the cytotoxicity study, the mitochondrial level reduction of the bromide salt of 3 (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT) to a colored product (formazan) was used ( Mossman T. J Immun Methods 1983, 65: 55-63). Peritoneal macrophages obtained from male Swiss mice were used. The cells were exposed to formulations containing hypotonic aqueous solutions of base liposomes. As a negative control, culture medium was used and as a positive control 0.005% benzalkonium chloride. The solutions were incubated at 37 ° C for 1 and 4 hours. The results obtained demonstrated an optimal tolerance for the base liposomes with and without vitamin E (Figures 3 and 4).

Brief description of the figures

Figure 1: Influence of the sonication process in the final size of the vesicles, comparing the use of an ultrasound probe for 2.5 minutes (- \ blacklozenge -) and an ultrasonic bath for 15 minutes (- \ blacksquare -). The frequency of each class, expressed as a percentage, is represented, versus the average size of it in \ mum.

Figure 2: Surface tension of the dispersion aqueous liposome (mN / m) depending on its concentration. The liposome concentration in the solution is expressed in phosphatidylcholine concentration (mM).

Figure 3: Cell viability (%) with hypotonic aqueous solutions of base liposomes with 100 and without vitamin E 101 incubated at 37 ° C for 1 hour. Two concentrations of liposomes (20 and 40 mg / ml) and two controls, one positive (0.005% benzalkonium chloride) and the other negative (culture medium) are studied.

Figure 4: Cell viability (%) with hypotonic aqueous solutions of base liposomes with 100 and without vitamin E 101 incubated at 37 ° C for 4 hours. Two concentrations of liposomes (20 and 40 mg / ml) and two controls, one positive (0.005% benzalkonium chloride) and the other negative (culture medium) are studied.

Claims (21)

1. Ophthalmic composition intended to act as a substitute for the precorneal film, characterized by containing liposomal vesicles of neutral or negatively charged phospholipids such as hydrophilic polar phase and non-polar lipids, both vehiculized in aqueous solutions containing mucin or substances with properties similar to mucin or mucoadhesive polymers. 2. The ophthalmic composition of the claim 1 containing mucoadhesive polymers such as hyaluronic acid, cellulose derivatives, chondroitin sulfate, chitosan, acid colominic, thiol derivatives (or other similar component). 3. The ophthalmic composition of the claim 1 containing a substance with mucomimetic properties. 4. The ophthalmic composition of the claim 1 containing neutral lipids and low polarity lipids such as waxes, cholesterol esters, triglycerides, free fatty acids and hydrocarbons. 5. The ophthalmic composition of the claim 1 containing lipocalins. 6. The ophthalmic composition of the claim 1 containing vitamin A. 7. The ophthalmic composition of the claim 1 containing sodium, potassium, calcium, chlorine and baking soda. 8. The ophthalmic composition of the claim 1 containing vitamin C. 9. The ophthalmic composition of the claim 1 containing lactoferrin. 10. The ophthalmic composition of the claim 1 containing albumin or pre-albumin 11. The ophthalmic composition of the claim 1 containing immunoglobulin A (IGA). 12. The ophthalmic composition of the claim 1 containing epithelial growth factor (epithelial growth factor: EGF). 13. The ophthalmic composition of the claim 1 containing transforming growth factor Beta (Beta transforming growth factor) (TGF-?). 14. The ophthalmic composition of the claim 1 containing fibroblast growth factor acid (Acidic fibroblast growth factor) (aFGF). 15. The ophthalmic composition of the claim 1 containing fibroblast growth factor basic. (Basic fibroblast growth factor) (bFGF). 16. The ophthalmic composition of the claim 1 containing antiproteases such as the macroglobulin 17. The ophthalmic composition of the claim 1 containing neural factors such as substance P e insulinlike growth factor (insulinlike growth factor). 18. The ophthalmic composition of the claim 1 containing antibacterial agents such as Ig G, lysozyme and complement. 19. The ophthalmic composition of the claim 1 containing long chain fatty acids such as gadoleic, palmitic, palmitoleic, stearic, oleic acid, linoleic, arachidic, linolenic, eicosenic, lignoceric, lactic and mystical. 20. The ophthalmic composition of the claim 1 containing hydrophilic lipids as phospholipids, sphingomyelin, ceramides and cerebrosides. 21. Use of the claimed composition for Prepare a medicine for the treatment of dry eye.